The insulin-like growth factors, IGF-I and IGF-II, are polypeptide growth factors that are chemically related to insulin. They are synthesized in many tissues, circulate in plasma, and bind to IGF-I receptors on target cells to stimulate cell growth, survival or differentiation. The biological activities of the IGFs are modulated by their interactions with a family of soluble proteins, the IGF binding proteins, to which they bind with high affinity. Typically, IGF:IGFBP complexes can not bind to signaling IGF-I receptors and are biologically inactive. The IGFBPs also may regulate cell growth independent of binding IGFs. We have studied the regulation of IGFBP-1 gene expression by insulin, and IGF-independent growth inhibition by IGFBP-3.(i) IGFBP-1 transcription is dynamically regulated in response to metabolic and hormonal changes. It is increased in diabetic rat liver, and rapidly normalized by insulin treatment. Insulin also inhibits IGFBP-1 transcription and IGFBP-1 promoter activity in the H4IIE rat hepatoma cell line. An insulin response element (IRE) has been identified in the proximal IGFBP-1 promoter. The signal transduction pathway that mediates insulin inhibition includes phosphatidylinositol 3-phosphate kinase (PI-3 kinase) and protein kinase B/Akt. Recent studies have shown that the transcription factor, forkhead rhabdomyosarcoma (FKHR), binds to the IRE to stimulate promoter activity. Insulin treatment results in phosphorylation of FKHR and a decrease in promoter activity. We have shown that cotransfection of mouse FKHR stimulates rat IGFBP-1 promoter activity in an insulin-inhibitable manner. A C-terminal fragment of mFKHR that contains the transactivation domain stimulates promoter activity and is inhibited by insulin, indicating that this fragment contains the information necessary for insulin inhibition. Insulin inhibition of promoter activity by the fragment was abolished by inhibitors of PI-3 kinase. Constitutively active PKB/Akt, the best characterized effector of PI-3 kinase, mimics insulin inhibition of promoter activity stimulated by full-length FKHR, but does not inhibit promoter activity stimulated by the C-terminal FKHR fragment. Neither of the two consensus sites for phosphorylation by protein kinase B/Akt that are present in the fragment is required for insulin inhibition. These results suggest that insulin regulation of the C-terminal FKHR fragment is dependent on PI-3 kinase but not on PKB/Akt, and may involve the phosphorylation of sites other than the PKB/Akt consensus sites. Studies are in progress to resolve whether insulin inhibition results from a direct effect on transcription. a redistribution of FKHR from the nucleus to the cytoplasm, or a combination of both mechanisms. (ii) IGFBP-3 has been reported to inhibit cell growth directly, as well as by forming biologically inactive complexes with IGF-I and IGF-II. It has been suggested that IGFBP-3 might mediate growth inhibitory effects of agents that increase IGFBP-3 expression, such as transforming growth factor-beta, anti-estrogens, and the tumor suppressor, p53. We have characterized growth inhibition by IGFBP-3 in CCL64 mink lung epithelial cells, and examined whether IGFBPs are involved in the inhibition of DNA synthesis by TGF-beta. Recombinant nonglycosylated human IGFBP-3 inhibits DNA synthesis in CCL64 cells. This is a direct effect of free IGFBP-3, since CCL64 cells do not synthesize detectable IGF-I or IGF-II. By contrast, no inhibition was observed with 1:1 molar complexes of IGFBP-3 and an IGF-I analogue, Leu60-IGF-I, that has low affinity for the IGF-I receptor. Direct growth inhibition by free IGFBP-3 may be mediated by a growth inhibitory IGFBP-3 receptor. 125I-labeled IGFBP-3 was crosslinked predominantly to an ~400-kDa protein on the CCL64 cell surface. Consistent with the ~400-kDa protein being the growth inhibitory IGFBP-3 receptor, binding was inhibited by coincubation with Leu60-IGF-I, mirroring the ability of Leu60-IGF-I:IGFBP-3 to abrogate the inhibition of DNA synthesis by IGFBP-3. TGF-beta also inhibits DNA synthesis in CCL64 cells. Surprisingly, TGF-beta inhibition also was substantially decreased by coincubation with Leu60-IGF-I. Since the only known action of Leu60-IGF-I is binding to IGFBPs, this result suggests that IGFBPs may be involved in the inhibition of CCL64 DNA synthesis by TGF-beta. The IGFBP synthesized by CCL64 cells was identified as IGFBP-2 by ligand blotting, immunoprecipitation and immunodepletion experiments. Consistent with a possible role for IGFBP-2 in mediating TGF-beta inhibition, recombinant bovine IGFBP-2 inhibited DNA synthesis, and inhibition was blocked by coincubation with Leu60-IGF-I. bIGFBP-2 does not bind to the ~400-kDa IGFBP-3 receptor, suggesting that it may act by a different mechanism than IGFBP-3. The relationship between TGF-beta and endogenous IGFBP-2 is unknown. Incubation with TGF-beta does not change the abundance of IGFBP-2 in CCL64 conditioned media. We propose that TGF-beta sensitizes cells to the growth inhibitory activity of endogenous IGFBP-2. Studies are in progress to define the mechanism for IGFBP-2 inhibition, and how it is affected by TGF-beta signaling.